1. Field of the Invention
The present invention generally relates to a codon-optimized nucleic acid for coding apo-clytin-II protein, apo-clytin-II protein, and method for preparing the same.
2. Description of Related Art
In the luminescent jellyfish of coelenterates, six photoproteins are now known to emit light by specifically binding to calcium ions. Among these proteins, aequorin and obelin have been studied in detail. These calcium-binding photoproteins have been used as a biological calcium indicator and for determining calcium concentration in cells. Furthermore, these proteins have been used in the following process. By using the gene of the protein, information about the important modifier and the biological mechanism in the cellular environment that are dependent upon calcium is deduced.
The photoprotein clytin was isolated from other luminous jellyfish, Clytia gregaria, in 1982 (see Non-patent Reference 1). Its gene was isolated in 1993, and it was confirmed that the gene could be expressed in E. coli (see Non-patent Reference 2). Thereafter, gene analysis was performed in detail by Inouye et al., and new gene was identified as new clytin with different luminescence property. The new gene from Clytia gregaria was named as clytin-II to distinguish from the previously isolated clytin (=clytin-I) (see Non-patent Reference 3). The clytin-II gene can be expressed in E. coli, and can be purified as recombinant clytin-II, which exhibits a potential application in the determination of a biological calcium indicator or the calcium concentration.
Compared with aequorin and clytin-I, the luminescence capacity of the total light emission per protein of clytin-II is almost the same; however, the initial luminescence intensity of clytin-II is about five times higher. Thus, the S/N ratio of clytin-II is five times higher than that of aequorin and clytin-I, which indicates that clytin-II may be superior to aequorin and clytin-I when used in a detection system.
Clytin-II having the luminescence ability is a complex containing apo-clytin-II protein with a molecular weight of about 22,000 and coelenterazine binding molecular oxygen. A molecule of the clytin-II protein has three sequences that can bind calcium ion. When clytin-II is bound to calcium ion, blue light (λmax=470 nm) is emitted, and carbon dioxide and coelenteramide are generated. Since the luminescence intensity is dependent on the concentration of calcium ion, clytin-II can be used to determine the calcium concentration with high sensitivity.
According to the prior art, the amino acid sequence of the photoprotein clytin-II is initially published by Inouye (see Non-patent Reference 3), and is disclosed in the EMBL sequence database under accession number AB360785 (disclosed as SEQ ID NO: 1 in this specification).
Amino acid sequence identity between clytin-II and other photoproteins, clytin-I, aequorin, obelin, and mitrocomin, is 88.4%, 61.9%, 76.2%, 60.8% respectively, while the identity with aequorin, obelin, and mitrocomin is low (see Non-patent Reference 3).
Furthermore, in Japanese Patent Publication 2005-506053, a codon-optimized (humanized) gene sequence based on the amino acid sequence of the photoprotein aequorin is reported. The amino acid sequence homology of codon-optimized aequorin with clytin-II shows 61.9% identity, and the luminescence pattern is different from clytin-II. Further, the nucleotide sequence identity between aequorin and clytin-II is 64.6%, so aequorin and clytin-II can be identified as totally different functional molecules.